PROJECT ABSTRACT Flaviviruses and alphaviruses are the two most common genera of arboviral pathogens, accounting for hundreds of millions of infections annually and repeated epidemics over the past 20 years. In total, these large genera include over a hundred potential human pathogens. Symptomatic infections with the flaviviruses (e.g. dengue, Zika, West Nile) and alphaviruses (e.g. chikungunya, Mayaro) present with non-specific manifestations and predominantly result in two overlapping acute clinical syndromes: a systemic illness with or without fever and a neurological illness such as meningoencephalitis. However, it is costly and impractical to test patients for more than a few common pathogens, and research protocols typically focus on one clinical syndrome. These factors complicate the detection of arboviral infections, surveillance and control efforts, and the calculation of accurate incidence estimates. Research in this proposal will utilize novel molecular methods for pan-genus detection and new multiplex serological methods to simultaneously screen for antibodies against eight arboviruses. The objective of the proposal is to evaluate new molecular and serological methods for the characterization of the incidence, seroprevalence and diversity of flavivirus and alphavirus infections among symptomatic patients. Research will be performed in Asuncin and the Central Department of Paraguay, where recent transmission of multiple arboviruses has been documented. Our central hypotheses are that 1) these new methods will increase the breadth of arbovirus detection among symptomatic patients, 2) evidence of exposure to multiple arboviruses will be common, and 3) broad, heterotypic antibody responses will protect against severe disease. This research will first characterize flavivirus and alphavirus infections among patients with an acute suspected arboviral illness or acute neurological disease (meningitis, encephalitis, Guillain-Barr syndrome). Pan-flavivirus and pan-alphavirus detection will be performed with a novel molecular assay. Viral epidemiology, clinical findings, and patient outcomes will be described among patients who meet the study definitions. The incidence of symptomatic and severe disease will then be determined in relation to arboviral antibodies at presentation. Multiplex serological testing will be performed to detect IgM and IgG against five flaviviruses and three alphaviruses. Seroprevalence will be calculated, and the incidence of symptomatic and severe disease will be evaluated in relation to the presence of pre-infection antibodies to heterotypic viruses. Finally, whole-genome viral sequences will be obtained directly from clinical samples using a CRISPR/CAS9 depletion system to analyze viral phylogenetics and identify new/rare viral strains. The purpose of this research is to improve laboratory methods for the most common human arbovirus families and identify patients at risk for symptomatic or severe arboviral illnesses. This work is expected to have an important positive impact because improved surveillance methods and patient risk stratification will result in increased arbovirus detection and targeted interventions for clinical management and outbreak control.